Bacillus cereus enterotoxin and method of use

ABSTRACT

The characterization of the protein structure of an enterotoxin to Bacillus cereus, designated hemolysin BL, is described herein. A high-yield purification scheme which allows quantitative characterization of the hemolysin BL activity is also described.

REFERENCE TO GOVERNMENT GRANT

This invention was made with United States Government support awarded bythe USDA, Grant # USDA 93-37201-9194. The United States Government hascertain rights in this invention.

FIELD OF THE INVENTION

The present invention is directed to the characterization of the proteinstructure, designated hemolysin BL, of Bacillus cereus and to ahigh-yield purification scheme which allows quantitativecharacterization of the hemolysin BL activity.

REFERENCE TO CITATIONS

A full bibliographic citation of the references cited in thisapplication can be found in the section preceding the claims.

DESCRIPTION OF THE PRIOR ART

Bacillus cereus (B. cereus) is a ubiquitous, usually commensal, soilorganism, but it is increasingly being recognized as a potentialpathogen, most commonly associated with food poisoning. It produces twodistinct food poisoning syndromes, characterized primarily by emesis anddiarrhea respectively (Kramer and Gilbert, 1989, Turnbull, 1986). Italso causes a variety of nongastrointestinal infections includingdevastating endophthalmitis, abscess formation, bacteremia, septicemia,cellulitis, endocarditis, meningitis, kidney and urinary tractinfections, osteomyelitis, puerperal sepsis and pulmonary and woundinfections (Turnbull, 1986).

The molecular nature of the virulence of B. cereus is poorly understood.However, the ability to produce dermonecrotic vascular permeabilityactivity correlates with the ability to cause diarrheal food poisoningTurnbull et al., 1979b) and nongastrointestinal B. cereus infections(Turnbull et al., 1979a, Turnbull et al., 1979b).

Thompson et al. (1984) described a dermonecrotic enterotoxigenic factorfrom B. cereus B-4ac. This toxin consisted of two or three distinctprotein components that were not active individually. The combinedcomponents were hemolytic, lethal to mice, cytotoxic to Vero cells, andpositive in ileal loop and vascular permeability tests. This toxin wasonly partially purified so its characterization was incomplete. Bitsaevand Ezepchuk (1987) have also reported a tripartite enterotoxic factorcalled DL-toxin (Bitsaev and Ezepchuk, 1987).

Beecher and Macmillan (1990, 1991) reported the purification of atripartite hemolysin, designated hemolysin BL, that was immunologicallyrelated to the multicomponent enterotoxin purified by Thompson et al.(1984) and exhibited vascular permeability activity. The hemolysin BLcomponents are designated B, L₁, and L₂. None of the components hashemolytic or VP activity individually and maximal activity occurs in thepresence of all three components. The complete nucleotide sequence ofthe gene encoding the B component has been determined (Heinrichs et al.,1993).

Isolated B component binds to erythrocytes and sensitizes them to thelytic action of the L components. The L components do not sensitizeerythrocytes to lytic action by B, and it is not yet known whether theybind to the cell membrane in the presence or absence of B. WhenB-sensitized erythrocytes are treated with either L₁ or L₂ and thenwashed, the cells lyse upon addition of the third component. HemolysinBL produces a characteristic and peculiar discontinuous pattern ofhemolysis on gel diffusion assays. When components are added together toa well in blood agar, hemolysis does not begin immediately next to thewell. Rather, after a period of diffusion, hemolysis begins at somedistance (typically several millimeters) from the well edge, forming aring around the well. With time, cells within the ring are completelylysed but hemolysis does not occur much beyond the initial diameter ofthe ring. Hemolysin BL was previously purified by anion exchangechromatography and preparative electrophoresis, using monoclonalantibodies to detect B and L₂. The yield was very low and onlyqualitative determinations of hemolytic and VP activities were possible.

There are currently two commercial immunoassays available for thedetection of the diarrheal enterotoxin of B. cereus; the BacillusDiarrhoeal Enterotoxin (BDE) Visual Immunoassay from Tecra, and the B.cereus Enterotoxin-Reverse Passive Latex Agglutination (BCET-RPLA TD950)kit from Oxoid. Both kits are in general use (Buchanan and Schultz,1992; Carlson et al, 1994; Granum et al, 1993). However, the value ofthese kits is questionable because the diarrheal enterotoxin has nowbeen identified as a three-component structure. Further confusing theissue, these kits detect different antigens (Christiansson, 1993).

SUMMARY OF THE INVENTION

The present invention is directed to a substantially pure proteincomposition "B" isolatable from Bacillus cereus and having the followingproperties: a) isoelectric point: 5.34; b) molecular weight (kD):37,800; c) N-terminal sequence: S-E-I-E-Q-T-N-N-G-T-A-L SEQ. ID. NO. 1!;d) extinction coefficient at 280nm, 1 cm path length: 1.32.

The present invention is also directed to a substantially pure proteincomposition "L₁ " isolatable from Bacillus cereus and having thefollowing properties: a) isoelectric point: 5.33; b) molecular weight(kD): 38,500; c) N-terminal sequence:x-E-T-I-A-Q-E-Q-K-V-G-N-Y-A-L-G-P-E, where x is undetermined SEQ. ID.NO. 2!; d) extinction coefficient at 280nm, 1 cm path length: 1.85.

The present invention is further directed to a substantially pureprotein composition "L₂ " isolatable from Bacillus cereus and having thefollowing properties: a) isoelectric point: 5.33; b) molecular weight(kD): 43,200; c) N-terminal sequence: E-T-Q-x-E-N-M-D-I-x-S, where x isundetermined SEQ. ID. NO. 3!, where x is undetermined; d) extinctioncoefficient at 280nm, 1 cm path length: 0.83.

The present invention is also directed to a substantially pureenterotoxin of Bacillus cereus comprising the following proteincompositions having the following properties: (B): a) isoelectric point:5.33; b) molecular weight (kD): 38,500; c) N-terminal sequence:S-E-I-E-Q-T-N-N-G-T-A-L SEQ. ID. NO. 1!; d) extinction coefficient at280nm, 1 cm path length: 1.32; (L₁): a) isoelectric point: 5.33; b)molecular weight (kD) : 38,500; c) N-terminal sequence:x-E-T-I-A-Q-E-Q-K-V-G-N-Y-A-L-G-P-E, where x is undetermined SEQ. ID.NO. 2!; d) extinction coefficient at 280nm, 1 cm path length: 1.85; (L₂): a) isoelectric point: 5.33; b) molecular weight (kD) : 43,200; c)N-terminal sequence: E-T-Q-x-E-N-M-D-I-x-S, where x is undetermined SEQ.ID. NO. 3!; d) extinction coefficient at 280nm, l1 cm path length: 0.83.

The present invention is also directed to a bioreagent suitable forantibody assays comprising a substantially pure protein compositionhaving the properties expressed in the previous paragraph.

The present invention is further directed to an antibody specific to theenterotoxin hemolysin BL.

Further, the present invention is directed to an antibody specific tohemolysin BL, said antibody being characterized in that it reacts withat least one of the three component proteins B, L₁ or L₂ of hemolysinBL.

The present invention is further directed to a diagnostic kit forassaying the presence of hemolysin BL comprising the antibody describedabove in one or more containers and directions for its use.

The present invention is also directed to an immunoassay method for thedetection of hemolysin BL, which comprises: contacting a samplesuspected of containing hemolysin BL with an antibody that is specificto hemolysin BL in order to form an immune complex, and determining thepresence of the complex in order to detect hemolysin BL in the sample.

Further, the present invention is directed to a process for isolatingprotein components of hemolysin BL from Bacillus cereus cells,comprising:

a. cultivating Bacillus cereus cells in an enriched medium containingthe nutritional substances necessary to grow and support the cells;

b. separating the cells from the medium;

c. concentrating the cells; and

d. separating the protein components.

Further still, the present invention is directed to an improved processfor isolating protein components of hemolysin BL from Bacillus cereuscells, the improvement comprising separating the protein components byhydroxylapatite chromatography.

The high-yield purification scheme which was developed in the presentinvention, allows quantitative characterization of hemolysin BL activityand determination of the molecular weight (MW), isoelectric point (pI),N-terminal sequence of each component, and extinction coefficients.Milligram quantities of the B, L₁, and L₂ components were highlypurified by a combination of anion exchange and hydroxylapatitechromatography.

A description of "extinction coefficient" is as follows: the amount thata protein absorbs at 280nm is a function of the tryptophan and tyrosineresidues present. Thus, the extinction coefficient (E) is an indicatorof the protein composition with respect to these two amino acids. Asused herein, the term "extinction coefficient" or "E" is defined as theabsorbance of 280nm of a 1 mg/ml protein solution through a 1 cm pathlength. To obtain the concentration of a protein, the absorbance at280nm (A₂₈₀) is divided by E for that protein. Reference is made toStoscheck (1990), which is incorporated herein by reference, for andescription of the determination of extinction coefficients.

The purification process of the present invention advantageouslyseparates and purifies the three components in hemolysin BL.

With this high-level separation, the hemolytic and vascular permeability(VP) activities of this toxin were characterized quantitatively. Adermonecrotic VP factor is believed to be a virulence factor in suchillnesses as diarrheal food poisoning as well as a variety of mild tosevere infections.

Estimation of the physical characteristics of the components is alsoimproved, and the amino-terminal sequence can now be determined for eachcomponent. Turbidimetric hemolysis assays of the toxin exhibits a highlyunusual dose response "zone phenomenon" that provides a tentative modelfor the mechanism of the discontinuous hemolytic pattern produced inblood agar. Hemolysin BL produced by the purification process of thepresent invention produced a severe dermonecrotic reaction that had notpreviously been observed. In addition, hemolysin BL is a diarrhealenterotoxin and may also be a major virulence factor in othernongastrointestinal infections.

The combined components had VP activity at low doses and were necroticat higher doses. The toxin exhibited an unusual dose response "zonephenomenon" in turbidimetric hemolysis assays. Activity increased up to200 ng/ml, then decreased to 350 ng/ml, and was constant up to at least2500 ng/ml. This behavior may provide an explanation for the unusualdiscontinuous pattern typical of hemolysin BL in gel diffusion assays.At high concentrations of one or two components the presence of lowamounts of the complementary component(s) resulted in full hemolyticactivity. Erythrocytes were protected from lysis by Zn++ at micromolarconcentrations but not by Ca++ and Mg++ up to 25 mM. These data provideguidelines for future work on this toxin and indicate that hemolysin BLis the dermonecrotic vascular permeability factor implicated as a B.cereus virulence factor.

The present invention is specifically useful to both regulatory agenciesand food industries and analytical laboratories in monitoring safety offood products carrying out epidemiologic studies, and managing foodpoisoning caused by B. cereus.

Other objects and advantages of the invention will be apparent from thefollowing detailed description and figures setting forth the preferredembodiment of the invention.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is an elution profile of hemolysin BL components from DE-52column in Experiment 1. (A) NaCl gradient and elution of protein asmeasured by A₂₈₀ ; (B) discontinuous () and continuous (▪) hemolysisactivity in gel diffusion assay. L activity (▴) was determined in thegel diffusion assay by complementation with B.

FIG. 2 is an SDS-PAGE of fractions from DE-52 chromatography inExperiment 1. Lanes: S, Pharmacia low-molecular-weight standards (sizesindicated on the left); P, ammonium sulfate-precipitated proteins fromculture supernatant of B. cereus F837/76. The numbers at the top of theother lanes correspond to the fraction numbers from the DE-52 column inFIG. 1. The top arrow in lane 12 points to L₂, the bottom arrow pointsto L₁, and the arrow in lane 26 points to B.

FIG. 3 are elution profiles of hemolysin BL components from an HA columnin Experiment 1. (A) The column was loaded with pooled fractions fromDE-52 column determined to contain the B component of hemolysin BL(fractions 24 to 29; FIG. 1 and 2.) (B) The column was loaded withpooled fractions from the DE-52 column determined to contain the L₁ andL₂ components of hemolysin BL (fractions 9 to 15; FIG. 1 and 2).

FIG. 4 is an SDS-PAGE of purified hemolysin BL components. Proteins wereeluted from a Mono-Q column. Lane S indicates the Pharmacialow-molecular-weight standards (sizes shown on left). The other lanesare labeled with the designations assigned to the respective componentsof hemolysin BL. The sample lanes were loaded with 200 ng of protein.

FIG. 5 is a graph illustrating dose-response curves in turbidimetrichemolysis assays for combined and individual components of hemolysin BLin Experiment 4. The curves were obtained by varying the concentrationof one or more components. The values along each x-axis represent thefinal concentration of the individual components that vary in thatexperiment. A: All three components were combined and the mixture wasdiluted to the indicated concentrations. B symbols: , the B componentwas held at 1000 ng/ml and each L component varied as indicated. ▪, bothL components were held at 1000 ng/ml and B varied as indicated. Csymbols: , B and L₂ were each held at 1000 ng/ml and L₁ varied asindicated. ▪, B and L₁ were held at 1000 ng/ml and L₂ varied asindicated. D symbols: , B was held at 100 ng/ml and L₁ and L₂ eachvaried as indicated. ▪, L₁ and L₂ were each held at 100 ng/ml and Bvaried as indicated. Hemolysis activity was determined after 1 h 15 minfor experiment A and 1 h 30 min for the other experiments.

DETAILED DESCRIPTION OF THE INVENTION

AMINO ACIDS

The amino acids are shown either by three letter or one letterabbreviations as follows:

    ______________________________________                                        Abbreviated Designation                                                                             Amino Acid                                              ______________________________________                                        A Ala                 Alanine                                                 C Cys                 Cysteine                                                D Asp                 Aspartic acid                                           E Glu                 Glutamic acid                                           F Phe                 Phenylalanine                                           G Gly                 Glycine                                                 H His                 Histidine                                               I Ile                 Isoleucine                                              K Lys                 Lysine                                                  L Leu                 Leucine                                                 M Met                 Methionine                                              N Asn                 Asparagine                                              P Pro                 Proline                                                 Q Gln                 Glutamine                                               R Arg                 Arginine                                                S Ser                 Serine                                                  T Thr                 Threonine                                               V Val                 Valine                                                  W Trp                 Tryptophan                                              Y Tyr                 Tyrosine                                                ______________________________________                                    

HEMOLYSIN BL

The present invention provides a process of isolating and purifyingprotein components of a B. cereus enterotoxin called hemolysin BL. Thistoxin is composed of three distinct proteins, designated B, L₁ and L₂with respective sizes of 37.7, 38.5 and 43.2 kDa and pIs of 5.34, 5.33and 5.33. The N-terminal amino acid sequences have been determined andare listed in Table 1, infra.

Hemolysin BL is a tripartite toxin with potent hemolytic, cytotoxic,dermonecrotic, and vascular permeability activities. The individualcomponents do not exhibit toxic activity individually, and maximalactivity requires all three components. Hemolysin BL is lytic in varyingdegrees to erythrocytes from different species, toxic to Chinese hamsterovary cells in culture, and retinal tissue in vitro and in vivo.

Hemolysin BL causes vascular permeability (VP) and dermonecrosis inrabbit skin, and fluid accumulation in rabbit ileal loops, whichestablishes hemolysin BL as a diarrheal enterotoxin.

PRODUCTION AND PURIFICATION OF ENTEROTOXIN/HEMOLYSIN BL

The present invention is directed specifically to a simple, gentle, highyield method of purifying the components of the tripartite hemolysin,designated hemolysin BL, which does not require access to specificantibodies. This purification scheme enables the improvement of thecharacterization of the individual hemolysin BL components.

The purification scheme provides ample antigen for antibody production,which allows development of specific hemolysin BL detection methods.Highly specific polyclonal and monoclonal antibodies have been producedand used to develop rapid and specific immunoassays for hemolysin BL.

The three components in hemolysin BL can be highly purified by ahigh-yield combination of anion exchange and hydroxylapatitechromatography. The present invention demonstrates a straightforward,high yield, chromatographic method for the purification of all threecomponents.

Growth medium:

The enterotoxin/hemolysin BL is secreted from B. cereus cells in the midto late log growth phase. The cells are grown in an appropriate mediumknown to the art for providing sufficient nutrients to allow rapidgrowth and toxin secretion. The most commonly used medium for toxinproduction in B. cereus is brain heart infusion broth supplemented with0.1% glucose (BHIG). Other media may be adapted for the purpose as well.These include any rich liquid media that promote rapid growth such as,but not limited, to standard formulations of nutrient broth, Trypticasesoy broth and beef extract. The media may be modified from the standardformula to enhance toxin production, e.g. by the addition of a useablecarbohydrate such as glucose.

The preferred medium for the present invention is caseaminoacids/glucose/yeast extract (CGY) broth. It is a modification of amedium described by Glatz and Goepfert (1977) and consists of 2%caseamino acids, 0.4% glucose, 0.6% yeast extract, 0.2% (NH4)₂ SO₄, 1.4%K₂ HPO₄, 0.6% KH₂ PO₄, 0.1% Na citrate, and 0.2% MgSO₄ (Beecher andWong, 1994). This medium is preferred over BHIG because it promotessimilarly high toxin production levels, but contains less heterogeneouscolored components, which complicate subsequent purification procedures.

Growth parameters and bacterial strain:

Growth conditions can be varied depending on the specific goals of theworker. Generally the goal is to maximize toxin production and minimizetoxin degradation. Specific optimal parameters, which vary with thebacterial strain used for toxin production, may be empiricallydetermined. The method of inoculation of the production batch willgenerally be standardized to minimize batch to batch variation.

The toxin may be grown as a stationary or aerated culture, in flasks orother containers, or in fermenters. Toxin production can be monitored bybioassay or immunoassay if antibodies are available. Relevant bioassaysinclude, but are not limited to, the ligated rabbit ileal loop test, therabbit vascular permeability (VP) test, and the discontinuous hemolysisassay of Beecher and Wong (1994).

Toxin production varies from one B. cereus strain to another. Any strainthat produces sufficient amounts of toxin to suit the workers' needs maybe used. For the present invention, the preferred bacterial strain is B.cereus F837/76, which produces high levels of hemolysin BL andrelatively low levels of other hemolysins and enzymes, such asproteases.

The preferred inoculation method is the addition of an overnight activestationary culture of the bacterium, usually in the same culture mediumas the production medium. The preferred inoculum size is 0.5 to 1% ofthe volume of the production culture. The preferred growth parametersare as follows: 5 to 6 hours at 32° C., 200 rpm on a rotary shaker, inflasks containing medium at one forth to one fifth the maximum volume ofthe flask.

Harvest of toxin:

Toxin harvest involves separation of cells from the culture supernatant.This may be done in any way practicable, including filtration orprecipitation. The preferred method for this invention iscentrifugation. The culture is centrifuged at 8,000× g for 10 to 20 min,the supernatant collected, and the pellet of cells discarded. Prior tocentrifugation, ethylenediaminetetraacetic (EDTA) is added to aconcentration of 1 mM to inhibit the major protease of B. cereus F837/76and minimize proteolytic degradation of hemolysin BL. Other proteaseinhibitors may be needed for other bacterial strains if they producedifferent proteases. Batch sizes range from 1 to 8 liters, but may bescaled up or down as needed.

Concentration of toxin:

The toxin generally must be concentrated to a volume that is convenientto handle in subsequent purification steps. This may be done by anymeans known to the art and practicable, including, but not limitedto, 1) ultrafiltration, 2) adsorption to an appropriate matrix such asan ion exchanger or hydroxylapatite followed by desorption with anappropriate buffer, 3) precipitation with an appropriate solvent orsalt, followed by collection of precipitate and solubilization ofproteins in a minimum volume. Adsorption and precipitation methods maybe varied such that a primary separation of hemolysin BL proteins fromsome other proteins is effected. The preferred method for this inventionis precipitation with 80 to 85% ammonium sulfate at 0° to 4° C. forgreater than 2 h. The precipitate is collected by centrifugation at8,000× g, for 30 min. Proteins are solubilized with a minimum volume ofwater.

Preparation of toxin concentrate:

The toxin concentrate may have to be prepared for the subsequentpurification step. The specific preparation will vary, depending on thefirst purification step chosen and will generally involve changing thebuffer in which the toxin is solubilized. This may be done by dialysisor gel filtration or dilution into the desired buffer. For thisinvention, the preferred preparation is overnight dialysis of theconcentrate at 4° C. against 100 to 200 volumes of 25 mM bis-Tris-HCl, 1mM EDTA, pH 5.9, with two buffer changes.

Purification of hemolysin BL components:

Purification of the hemolysin BL components may include any combinationof methods that result in the separation to substantial purity of eachor any individual component from the other components and othernon-hemolysin BL proteins. Potentially useful methods include, but arenot limited to 1) column chromatography, including ion exchangechromatography, and chromatofocusing (a form of ion exchangechromatography in which proteins elute from the column near theirisoelectric points), 2) hydroxylapatite chromatography, 3) gelfiltration chromatography, 4) isoelectric focusing, 5) preparativeelectrophoresis, 6) gel filtration chromatography, which allows limitedprotein load for optimal resolution and permits only poor resolution ofproteins in the size range of the hemolysin BL components and the manyother proteins within that range, 7) isoelectric focusing, which allowslimited protein load and yield and presents numerous technicaldifficulties in its performance, and 8) preparative electrophoresis,which allows low protein load and yield.

Column chromatography using adsorptive matrices is preferred for thisinvention because of the high resolution, high load capacities andyields, and great variation of column sizes allowed by these methods.

Anion exchange chromatography is preferred over cation exchangechromatography because the pH required to bind the proteins comprisinghemolysin BL is 4 or lower, which is detrimental to the stability of thecomponents. Proteins bind to ion exchangers as a function of the surfacecharge of each at a given pH. Usually proteins bind to anion exchangersone pH unit above their pIs and to cation exchangers one pH unit belowtheir pIs.

Hydroxylapatite (HA) chromatography is preferred for this inventionbecause of unexpectedly and unusually good resolution of the componentsfrom one another. Proteins bind to HA as a function of surfaceproperties (largely surface charge). The hemolysin BL components elutefrom HA in an order unpredictable from their isoelectric points andbehavior on anion exchangers. L₁ and L₂ closely coelute from anionexchangers but may be well resolved from one another upon elution fromHA. The B component, which behaves on anion exchangers as if it has amore acidic surface than the L components, elutes very late from HA. Theelution of B with the relatively high phosphate concentration of around120 mM is suggestive of a neutral surface charge (pI 6 to 7) or ofspecific interaction with phosphate.

The preferred mode of elution of hemolysin BL components is with lineargradients of NaCl or phosphate from anion exchangers and HArespectively. Other methods may be used, such as step gradients of NaCland phosphate, or pH change, but resolution will be sacrificed.

The purification (chromatographic) steps may be applied in any orderthat results in substantially pure preparations of the components ofhemolysin BL. The preferred sequence for this invention is 1) anionexchange chromatography, 2) HA chromatography and 3) anion exchangechromatography.

Step 1 results in separation of component B from the two L componentsand minimal separation of L₁ from L₂. Fractions containing the proteinsof interest are identified by the methods described below andappropriate fractions are applied directly to the HA column. Usually,two pools of fractions containing respectively 1) B and 2) L₁ plus L₂are separately applied to and eluted from a HA column. The fractionsfrom step 1 (anion exchange) are applied directly to the HA column instep 2 without a change in the buffer composition of the samples.Ideally, only three chromatographic columns (one anion exchange and twoHA columns) are needed to complete steps 1 and 2, with no manipulationof sample buffer composition between the steps.

If the steps are reversed, i.e., HA first, followed by anion exchange,the hemolysin BL components will not bind directly to the anionexchanger after elution from HA. The pooled samples must be placed bydialysis or gel filtration into the anion exchange binding buffer, whichadds a manipulation step compared with the preferred sequence. If HA isused as step 1, the three hemolysin BL components will separate from oneanother, necessitating the collection of a minimum of three sample poolsfor step two. At minimum the HA followed by anion exchange sequence willresult in one HA column and three anion exchange columns. The actualnumber of column runs needed to produce substantially pure hemolysin BLcomponents will vary depending on the column sizes and protein loadsused. Overloading of columns results in decreased resolution. A remedyfor a given amount of protein on a given column is to apply less percolumn run, e.g. apply portions of sample pools, or collect multiplesmall sample pools.

The third chromatographic step is a second anion exchange column. Thisstep is included as a high resolution step to remove low levelcontaminants of other hemolysin BL components and other proteins fromthe component preparations. The step requires a change of buffer to theanion exchange binding buffer described above and below. This step isperformed on a small column that allows highly reproducible separationsof high resolution. Protein loads are determined by the capacity andresolving power of the column to be used. This step is optional and maybe omitted if the preparations from step two are of sufficient purity tosuit the specific needs of the worker.

Identification of proteins comprising hemolysin BL:

Hemolysin BL components in the chromatographic fraction may beidentified by their respective activities, or by immunoassay ifantibodies are available. The preferred method for this invention is thegel diffusion hemolysis assay described below, coupled withidentification of proteins of specific sizes on SDS-PAGE gels.

BIOREAGENTS

The protein components of hemolysin BL can be isolated for use asbioreagents and used to prepare polyclonal and monoclonal antibodies fordetection of the hemolysin BL enterotoxin. The antibodies can be used intest kits which are used to diagnose clinically suspected contaminationof hemolysin BL. The proteins can be recognized and distinguished fromother proteins according to the characteristics determined in theexperiments below.

PRODUCTION OF ANTIBODIES TO HEMOLYSIN BL

Antibodies can be prepared according to methods known to the art.Polyclonal antibodies can be produced by injecting electrophoresis gelslices containing separated hemolysin BL components into rabbitsessentially as described by Harlow and Lane (1988). Antisera to allthree hemolysin components react with a single major band from B. cereusculture supernatants on Western immunoblots and will exhibit reactionsof nonidentity with each other in double immunodiffusion assays.

Hemolysin BL proteins can also be employed to produce monoclonalantibodies to the proteins utilizing the procedure described by Fazekuset al. (1980). The essential steps are as follows:

1. immunize an animal, preferably a rodent such as a rat or mouse, or arabbit, with the protein component;

2. isolate β-lymphocytes, suitably spleen lymphocytes, from theimmunized animal;

3. fuse the isolated β-lymphocytes with myeloma cells from an animal,preferably a rodent such as a rat or mouse;

4. select from the fused cells those hybridoma cell lines which reactpositively with the proteins;

5. clone the hybridoma cells to produce additional monoclonalantibodies.

In general overview, polyclonal and monoclonal antibodies (referencedherein as "antibodies" unless otherwise specified) specific forhemolysin BL can be produced by immunizing BALB/c mice with hemolysinBL. Spleen cells from the mice so immunized are fused with a mousemyeloma cell, with the fusion being effected with treatment inpolyethylene glycol in accordance with known methods. The resultinghybridomas are cultured and then selected for antibody activity. Thecells producing antibodies specific to hemolysin BL are detected bymeans of an ELISA in the manner well known to the art. The antibody ispurified from ascites fluid in conventional ways. The cloned cell lineso created and selected is perpetuated by conventional cell culturingtechniques.

Any of a large number of clinical tests may be employed utilizing theantibodies of this invention. Typical tests include radioimmunoassay,enzyme-linked-immunoassay (ELISA), precipitation, agglutination, directand indirect immunofluorescence and complement fixation. These tests mayemploy competitive and sandwich-type assays.

The antibodies are tested for specificity by ELISAs and byimmunoblotting of a variety of enterics. By these means, it can bedetermined that the antibody forms a strong reaction by direct ELISAwith hemolysin BL.

ELISAs are a conventional method for assaying for the presence of anantigen in a sample of test material. The sandwich ELISA of theinvention is adapted to assay for the presence of hemolysin BL in asample of test material and includes the following steps. First, a knownantibody to hemolysin BL is bound to a suitable adsorbtor substrate.Preferably, a plastic culture plate is used, such as a 96-wellpolystyrene culture plate (Costar, Cambridge, Mass.--Model No. 3596). Asolution of antibody to hemolysin BL is placed in each of the wells andallowed to remain under conditions such that the antibody to hemolysinBL is adsorbed to the surface of the wells. Unadsorbed antibody solutionis then washed away, leaving the antibody to hemolysin BL bound to theadsorptive walls of the wells, which shall be referred to as "adsorbtorsubstrate units." With antibody to hemolysin BL adsorbed to them, theyshall be referred to as "antibody to hemolysin BL substrate units." Theantibody to hemolysin BL substrate units is then treated with anappropriate blocking reagent, such as nonfat dried milk, to blocknon-specific binding sites. After appropriate incubation, this reagentis removed.

Next, a known quantity of the test material is exposed to the antibodyto hemolysin BL-charged substrate units for an appropriate period oftime, and then is removed by washing. Any hemolysin BL in the testmaterial will bind to the antibody to charged substrate units.

Similarly, a standard preparation of hemolysin BL is exposed to anotherset of antibodies to hemolysin BL-charged substrate units to serve as acontrol.

The antibody referred to above is added to the antibody to hemolysinBL-charged substrate units to bind with any bound hemolysin BL. Afterappropriate incubation, the unbound antibody is removed by washing.

The antibody to hemolysin BL-charged substrate units are reacted withtest samples of hemolysin BL and are then assayed for the presence ofthe antibody.

Preferably this is done by exposing antibody to hemolysin BL-chargedsubstrate units reacted with the test samples or hemolysin BL and theantibody thereon to a marker-coupled anti-mouse antibody to allow themarker-coupled antibody to bind to any antibody present. The unboundmarker-coupled antibody is then removed, and the amount of markerremaining on the antibody to hemolysin BL-charged substrate units ismeasured. The marker may be an enzyme measured by its effect on aselected reagent, a fluorescent material, a radioactive material, or anyother of the markers familiar to one skilled in the art. It will beapparent that the antibody itself may be combined directly with amarker, whereupon the step of reacting a marker-coupled anti-mouseantibody may be omitted.

The antibody may also be used in other conventional ELISAs. For example,a sample of test material may be bound to an adsorbtor substrate andthen exposed to the antibody disclosed above. The antibody binds to anyhemolysin BL present in the test material. Unbound portions of theantibody are then removed. Next, an assay comparable to those discussedabove is conducted for the presence of bound antibody.

The antibody of the present invention may also be used in any of thegenerally known methods of using such antibodies in immunohistologicaltechniques for examining a substantially cohesive, nonfluid testmaterial, such as a cell or tissue sample. Preferably, the sample is afood product or a fecal sample or an extract of the same. The testmaterial is incubated with the antibody to bind the antibody tohemolysin BL present in the test material. The test material is thenwashed to remove the unbound portion of the antibody. The antibody maythen be reacted in such a way as to make its presence visually apparent.Typically, the test material bearing antibody bound to hemolysin BLcontained therein is incubated with a marker-labeled anti-mouse antibodycomparable to those discussed above. The marker-labeled antibody bindsto the antibody. A marker is selected such that it may be made visuallyapparent. Fluorescent and enzyme markers typically are used. The testmaterial is then microscopically observed under conditions adapted torender the marker visually perceivable. The antibody to hemolysin BL isspecifically useful as a reagent for the rapid detection of hemolysin BLin food and clinical specimens.

The present invention also includes kits, e.g., diagnostic assay kits,for utilizing the antibody to hemolysin BL and carrying out the methoddisclosed above. In one embodiment, the diagnostic kit wouldconventionally include the antibody to the hemolysin BL in one or morecontainers, a conjugate of a specific binding partner for the antibody,a label capable of producing a detectable signal, and instructions forits use. The antibody can be a polyclonal antibody, a monoclonalantibody, a mixture of polyclonal antibodies, a mixture of monoclonalantibodies, and a mixture of polyclonal and monoclonal antibodies. Thekit may be conjugated to a label, as is well known to the art. Variouslabels include enzymes, radioisotopes, particulate labels, chromogens,fluorescers, chemiluminescers, coenzymes, free radicals, andbacteriophages. Additionally the antibody may be bound to a support.

The instructions for use are suitable to enable an end user to carry outthe desired test. By the term "instructions for use," it is meant atangible expression describing the reagent concentration for at leastone assay method, parameters such as the relative amount of reagent andsample to be admixed, maintenance time periods for reagent/sampleadmixtures, temperature, buffer conditions and the like. It is withinthe scope of this invention to provide manual test kits or test kits foruse in automated analyzers.

A specific diagnostic kit could be in a dipstick format. This couldinvolve adsorbing polyclonal immunoglobulin to a hydrophobicpolyvinylidene difluoride (PVDF)-based membrane. The PVDF membrane isthen treated with 5% bovine serum albumin to block nonspecific bindingsites. The PVDF membrane is dipped for 30 minutes in an enrichmentculture of food that may contain hemolysin BL. After washing, the PVDFmembrane is treated with the antibody. This antibody will bind to anyhemolysin BL cells bound to the PVDF membrane. The bound antibody isdetected with alkaline phosphatase-conjugated goat anti-mouseimmunoglobulin that reacts with Nitro bluetetrazolium/5-bromo-4-chloro-3-indolylphosphate substrate to producepurple spots as a positive reaction.

Each of the protein components B, L₁ and L₂ can also be isolated by thepurification method described herein as a bioreagent and used to prepareantibodies for detection of hemolysin BL in a sample. The antibodies canbe provided in test kits which are used to diagnose cases of suspectedhemolysin contamination.

The examples below provide specific examples of the invention disclosedherein.

EXAMPLES

The following examples are presented to illustrate the advantages of thepresent invention and to assist one of ordinary skill in making andusing the same. The examples are not intended in any way to otherwiselimit the scope of the disclosure or protection granted by the patent.

Experiment 1

Purification of Hemolysin BL Culture Medium

The culture medium used for the production of hemolysin BL was amodification of one described by Glatz and Goepfert (1977). It consistsof 2% caseamino acids, 0.4% glucose, 0.6% yeast extract, 0.2% (NH₄)₂SO₄, 1.4% K2HPO₄, 0.6% KH₂ PO₄, 0.1% Na citrate, and 0.2% MgSO₄. We havefound that levels of hemolysin BL produced in this medium (termed "CGY")are comparable to those attained in brain heart infusion brothsupplemented with 0.1% glucose (BHIG), which is the most commonly usedmedium for B. cereus toxin production. However, CGY contains much lowerlevels of colored contaminants, making purification easier than withBHIG.

Production of hemolysin BL:

Production of hemolysin BL from B. cereus F837/76 was as describedpreviously (Beecher and Macmillan, 1990), except that cultures weregrown at 32° C. instead of 37° C. and EDTA (1 mM) was added at the timeof harvest (5 h) to inhibit proteolysis. Four liters of culturesupernatant was concentrated with 90% saturated (NH₄)₂ SO₄, resuspendedin 50 ml of water, and dialyzed overnight at 40° C. against 2 liters of25 mM Bis-Tris-HCl, 1 mM EDTA, pH 5.9, with two buffer changes.

Chromatography:

Whatman DE-52 was charged with counter ions with 0.25 M Bis-Tris-HCl, pH5.9, and packed into a 2.6 cm diameter column to a height of 9 cm. Thecolumn was equilibrated with 25 mM Bis-Tris-HCl, pH 5.9, at a flow rateof 3 ml/min. The sample was dialyzed against the equilibration bufferplus 1 mM EDTA prior to application to the anion exchanger.

Proteins were eluted with a linear gradient of NaCl from 0 to 0.5 M over400 ml and collected in 6-ml fractions. Hydroxylapatite (HA)chromatography was performed using a 1.6 by 6.4 cm column of Bio-Gel®HTP (BioRad, Richmond, Calif.) equilibrated with 10 mM NaCl. Selectedfractions from the DE-52 column were pooled and applied directly to theHA column at a flow rate of 1.5 ml/min. Proteins were eluted into 2.5 mlfractions with a linear gradient of sodium phosphate buffer, pH 6.8,from 0 to 0.24 M over 120 ml.

To concentrate proteins from HA chromatography, pooled fractions weredialyzed against 25 mM Bis-Tris-HCl, pH 5.9, and applied to a Mono-Q(MQ) HR 5/5 anion exchange column (0.5 by 5 cm, Pharmacia) equilibratedwith the same buffer. Proteins were eluted with a linear gradient ofNaCl from 0 to 0.25 M over 40 ml at a flow rate of 1 ml/min. This stepalso served as a high-resolution anion exchange step that removed minorcontaminating proteins.

Electrophoresis and protein blotting:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)with buffer formulations of Hames (1981) were run in a Mini Protean IIdual slab cell (BioRad).

The minigels were either silver-stained by the method of Blum et al.(1987) or used for protein blotting. Proteins resolved in SDS-PAGE gelswere transferred to polyvinylidine difluoride (PVDF) membranes(Millipore, Bedford, Mass.) in a Mini Trans-BlotR electrophoretictransfer cell (BioRad) at 100 V constant voltage for 1 h 15 min.Isoelectric points were determined on a PhastSystem (Pharmacia,Piscataway, N.J.) using Pharmacia IEF standards and PhastgelR IEF gelswith a pH range of 4 to 6.5.

Results:

Reference is made to FIG. 1, which represents the elution profile of B.cereus proteins from the DE-52 anion exchange column. The discontinuouslytic pattern typical of hemolysin BL in gel diffusion assays wasproduced directly by fractions 20 to 40. Production of the discontinuouspattern indicates the presence of all three hemolysin BL components. Onprevious DE-52 columns (Beecher and Macmillan, 1991) the B component waswell separated from the L components indicating that the activity seenin these fractions was the result of tailing of small concentrations ofthe L components into the peak fractions of the B component.

Fractions containing the L components did not possess discontinuoushemolytic activity and were located by complementation with the Bcomponent. Partially purified preparations of B that still possesseddiscontinuous hemolytic activity could be used for detection of Lactivity. A lytic zone eventually developed between the wells and beyondthe hemolytic zone of the B-containing fractions.

Some of the fractions containing L components possessed continuous typehemolytic activity, i.e., lysis began at the well edge and proceededoutward. We do not yet know if this activity is due to a differenthemolysin, the combination of sphingomyelinase and lecithinase (Gilmoreet al., 1989), or the combination of L components with other B. cereusmetabolites.

Based on the presence of proteins of appropriate size, SDS-PAGE analysisof the fractions from the DE-52 column (FIG. 2) confirmed that B waswell separated from the two L components, which coeluted. This gel wasused to determine which fractions to pool for the next chromatographicstep. Fractions 24 to 29 (containing the B component) were pooled andapplied to the HA column. Fractions containing B were identified asthose which produced complementary hemolysis with L-containing fractionsfrom the DE-52 column. B eluted under the large peak at approximately160 mM phosphate as illustrated in FIG. 3A.

For isolation of the L components, fractions 9 to 15 were pooled andapplied to the HA column. L₁ and L₂ eluted under the large peaks ataround 90 and 125 mM phosphate respectively as illustrated in FIG. 3B.The identities of the L components were established by SDS-PAGEanalysis. At this step all three components exhibited single bands onCoomassie Blue stained gels (not shown). The final chromatographic stepon the MQ anion exchange column resulted in highly purified preparationsof all three components as determined with an SDS-PAGE gel stained withsilver as illustrated in FIG. 4. Assuming that the absorbency at 280 nmrepresented the concentration of the purified component by the HA step,the yields of the components from 4 liters of culture supernatant were2.2, 2.0, and 2.9 mg of B, L₁, and L₂ respectively. At the MQ step theyields were 1.4, 0.9, and 1.8 mg respectively. Using subsequentlydetermined extinction coefficients, the yields were as follows for B: HA1.7mg; MQ 1.0 mg. The yields for L₁ were as follows: HA 1.0 mg; MQ 0.5mg. The yields for L₂ were as follows: HA 3.5 mg; MQ 2.2.

Experiment 2

Analysis of Amino-Terminal Amino Acids

Purified proteins were prepared for N-terminal sequence analysis asdescribed by Matsudaira (1990). Electroblotted proteins were cut fromthe transfer membranes and sent to the Protein/Nucleic Acid SharedFacility at the Cancer Center of the Medical College of Wisconsin forN-terminal sequence analysis by Edman degradation.

Physical characteristics of hemolysin BL components:

Table 1 lists experimentally determined physical characteristics of thecomponents of hemolysin BL. A single band was visible for all threecomponents by SDS-PAGE (FIG. 4) and for the B component by IEF. L₁ andL₂ each exhibited a second minor band by IEF of pI 5.20 and 5.26respectively. We do not yet know whether these minor bands representisoforms of the components or contaminants.

                                      TABLE 1                                     __________________________________________________________________________    Physical Characteristics of the Components of Hemolysin BL                          Mol                                                                     Component                                                                           wt.sup.a                                                                          pl.sup.b                                                                            N terminus.sup.c  E.sup.d                                                                          Seq ID#                                  __________________________________________________________________________    B     37,800                                                                            5.34 ± 0.012                                                                     S-E-I-E-Q-T-N-N-G-D-T-A-L                                                                       1.32                                                                             1                                        L.sub.1                                                                             38,500                                                                            5.33 ± 0.008                                                                     x-E-T-I-A-Q-E-Q-K-V-G-N-Y-A-L-G-P-E                                                             1.85                                                                             2                                        L.sub.2                                                                             43,200                                                                            5.33 ± 0.016                                                                     E-T-Q-x-E-N-M-D-I-x-S                                                                           0.83                                                                             3                                        __________________________________________________________________________     .sup.a Calculated from the SDSpolyacrylamide gel in FIG. 4                    .sup.b Average value for three different IEF gels                             .sup.c x, undetermined residue                                                .sup.d Extinction coefficient at 280 nm, 1 cm path length                

Experiment 3

Vascular Permeability Assay

Vascular permeability activity was assayed as described by Glatz et al.(1974). Rabbits of either 2-3 kg or 4-5 kg were used. The larger animalsproduced more distinct reactions. Data obtained from different rabbitsare reported separately. All activities are reported as averages ofduplicate samples.

The components of hemolysin BL were tested individually and incombination for vascular permeability activity in rabbit skin. In acutaneous edematous reaction, microvascular permeability is compromised,allowing leakage of serum proteins into the interstices with subsequentaccumulation of fluid. The reaction can be quantitated by theintravenous injection of Evans blue dye, which binds serum proteins andproduces an area of bluing surrounding the injection site of apermeability factor (Tu and Miller, 1992). At high concentrations, theB. cereus vascular permeability factor/enterotoxin produces a centralarea of necrosis within the larger areas of bluing and swelling (Glatzet al., 1974).

Results:

Reference is made to Table 2 which shows the VP activity of hemolysin BLcomponents individually and in combination:

                  TABLE 2                                                         ______________________________________                                        VP Activity of Hemolysin BL Components Individually and in                    Combination                                                                             Amt injected                                                        Component(s)                                                                            (μg)     Reaction diam (mm).sup.a                                injected  Each    Total   Edema.sup.b                                                                          Bluing Necrosis                              ______________________________________                                        B only    7.68    7.68    --.sup.c                                                                             --     --                                    L.sub.1 only                                                                            7.68    7.68    --     --     --                                    L.sub.2 only                                                                            7.86    7.68    --     --     --                                    All       2.56    7.68    NE.sup.d                                                                             19.0   12.0                                  All       1.28    3.84    NE     18.0   10.0                                  All       0.64    1.92    21.5   19.0   8.5                                   All       0.32    0.96    21.5   19.5   6.5                                   All       0.16    0.48    20.5   12.5   4.0                                   All       0.08    0.24    18.0   8.0    --                                    All       0.04    0.12    13.0   4.5    --                                    All       0.02    0.06    --     --     --                                    All       0.01    0.03    --     --     --                                    ______________________________________                                         .sup.a Average of duplicate samples in the same rabbit                        .sup.b Defined as the raised area surrounding an injection site               .sup.c --, no reaction visible                                                .sup.d NE, edema not seen for these samples. Lesions were flat areas of       necrosis surrounded by a ring of bluing.                                 

No dermal reaction was noted after injection of 2.5 ug of eachindividual component or in the combinations of B+L₂ and L₁ +L₂containing 2.5 ug of each component. The combination of B+L₁ produced 14mm of edema, 3 to 4 mm of bluing, and no necrosis. The combination ofall three components produced 13 mm of bluing and 11 mm of necrosis, butno edema. Necrotic activity was not previously seen for hemolysin BL.The edematous reaction of the B+L₁ combination was seen previously(Beecher and Macmillan, 1991). The enhancement of the necrotic andbluing responses by addition of L₂ is also consistent with previousobservations, but the lack of edema was surprising. The dose-responsedata presented in Table 2 suggest that diminished edema ischaracteristic of high concentrations of the toxin. Extensive tissuedestruction may prevent the retention of fluid, thereby reducingswelling.

In the experiment shown in Table 2, no response occurred with theinjection of 7.68 ug of each component individually. This quantity wasequal to the highest total protein content in the dose response series.Our observations also indicate that there is cooperative activitybetween B and L₁ and between B+L₁ and L₂.

The VP data presented here establish clearly that hemolysin BL is thenecrotic vascular permeability factor of B. cereus and that it iscomposed of three cooperatively acting proteins that have no activityindividually.

Experiment 4

Hemolysis Assays

This experiment was performed to estimate the best working concentrationrange for the components for specific studies. Gel diffusion assays forhemolysin BL were as described previously (Beecher and Macmillan, 1990;Beecher and Macmillan, 1991). Sheep blood (2.5 to 3% v/v) agar waspoured onto GelBondR film (FMC Bioproducts, Rockland, Me.) to 0.15ml/cm2 so that wells of 3 mm diameter would accommodate samples of 10ul.

Activity is reported as the distance from the well edge to the outeredge of the hemolysis zone, which was calculated by measuring the zonediameter and subtracting the radius of the well (1.5 mm) from the totalzone radius. For estimation of L activity, fractions were placed inwells adjacent to, and equidistant from, wells containing B.

After a period of diffusion (several hours to overnight) the lytic zonesbetween the wells were measured. Activity is expressed as the length inmillimeters of these lytic zones. This activity was arbitrarily chosento give a rough estimate of the level of L activity in fractions. Theactual level of each L component cannot presently be determined.Hemolysin BL activity was also determined with a microtiter plate assaysimilar to that described by Young et al. (1986) . In most experiments,the concentration of one or more of the reaction components was heldconstant in a series of wells while the other components were varied.Dilutions were prepared either in separate tubes or directly in themicrotiter wells. The varying components were added in 50 ul aliquots totheir respective wells at concentrations four times the desired finalconcentrations. The constant components were added in 50 ul aliquots toall wells, also at a concentration four times the final desiredconcentration.

When all components were varied, they were added to their respectivewells in 100 ul aliquots. Hemolysis was initiated by the addition of 100ul of sheep blood diluted in 50 mM Tris-HCl, 150 mM NaCl, pH 7.4 (TBS)to twice the desired final concentration. The final concentration wasone that had an optical density of 0.8 to 0.9 at 630 nm (ca. 2.5% v/v).The assay plate was held at 37° C. and the A630 determined at 15 minintervals. Activity was reported at a time point at which hemolysiswas >50% and <90% (generally 1.5 h). For 0% hemolysis controls, sampleswere 100 ul TBS and for 10.0% hemolysis controls, samples were 100 ul ofwater containing saponin (Bernheimer, 1988). Values for control sampleswere taken as averages for six replicates. All samples were intriplicate and the reported values are averages. The microplate readerwas blanked against a 100% hemolysis control and percent hemolysis wascalculated by the following formula: (A₆₃₀ negative control--A₆₃₀sample)/A₆₃₀ sample/A₆₃₀ negative control! X 100.

Dose responses of hemolysin BL components in turbidimetric hemolysisassays:

Reference is made to FIG. 5A which depicts a complex dose response curvecharacteristic of hemolysin BL. All three components were each presentat the concentrations indicated on the x-axis. There were two unusualfeatures to this curve. First, the activity plateaued above 350 ng/ml.In a separate experiment (not shown) this plateau was nearly flat up to2500 ng/ml (the maximum concentration tested). Second, there was aconcentration range, between 350 ng/ml and 100 ng/ml, in which activitywas greater than at higher concentrations.

The dose response curve for the combination of all three hemolysin BLcomponents (FIG. 5A) is unusual in two respects. First, the activity ofhemolytic agents does not generally plateau with increasingconcentration, particularly at such a low concentration (Ponder, 1948).Second, the occurrence of a concentration range (between 100 and 300ng/ml) in which hemolysis is greater than at higher concentrations,combined with the plateau, appears to be a unique phenomenon. Similar,but not identical phenomena have been observed for hemolysis by sodiumtaurocholate and by sodium glycocholate at pH extremes, and for acombination of colloidal silicic acid and complement (Ponder, 1948).

In the experiments depicted in FIGS. 5B-D, the concentrations of one ormore components were held constant and the other components were varied.When the constant component(s) was/were in excess there were significantdose responses only below 40 ng/ml (each component) for L₁ +L₂, 300ng/ml for B, 30 ng/ml for L₁, and 125 ng/ml for L₂ With B+L₁ each at1000 ng/ml there was 15.5% hemolysis in the absence of L₂ and B+L₂ alonecaused 10% lysis. None of the individual components, nor the combinationof L₁ +L₂, caused lysis up to at least 2500 ng/ml each.

These data suggest that, for working in a dose-dependent concentrationrange (e.g. when studying the effect of potential inhibitors), eachcomponent should be present below about 200 ng/ml. In FIG. 5D, B or L₁and L₂ were held at 100 ng/ml and there was a dose response for B and Lwithin the 200 ng/ml range.

Dose-responses of hemolysin BL components in gel diffusion assays:

The dose-response of hemolysin BL in gel diffusion assays was examined.The addition of 10 ng or 1000 ng of each component per well resulted inlytic radii of approximately 1 mm and 3.8 mm respectively. The increaseof lytic radius with concentration was asymptotic. Plotting the amountof hemolysin BL per well on a log10 scale produced a roughly linear doseresponse curve, suggesting that zone size may prove useful forquantitation of hemolysin BL. However, possible effects of variations inthe ratios of the components to one another have not been examined.

In another gel diffusion experiment the B component was held at 100ng/well while the amount of L₁ and L₂ was varied. Significant activitywas detected with 0.5 ng each of L₁ and L₂ per well. The lytic radiusincreased sharply to 2.5 ng/well and more gradually to 60 ng/well. Above60 ng of L₁ and L₂ per well there was no effect on the size of the lyticzone.

The concentration of blood in a series of gels was varied from 0.5 to10% and wells in each gel were each loaded with 100 ng of B and 50 ngeach of L₁ and L₂. The distance from the well edge to the edge of thelytic zone varied linearly from 2.2 mm in 10 blood to 3.4 mm in 0.5%blood.

Effect of divalent cations on hemolysis by hemolysin BL:

In turbidimetric assays of hemolysin BL there was a dose-dependentinhibition of hemolysis by Zn++ from 10 to 500 uM. Complete protectionwas seen for Zn++ concentrations of 500 uM and higher. EDTA (1 mM)prevented the protection of erythrocytes by Zn++. Erythrocytes were notprotected by Mg++ or Ca++ up to 25 mM. Complete hemolysis by hemolysinBL at 100 ng/ml typically took greater than 1.5 hours. When erythrocyteswere treated for 1.5 h with hemolysin BL (100 ng/ml per component) andZn++ (500 uM) no lysis occurred, but addition at that time of 1 mM EDTAcaused complete lysis in less than one minute.

Experiment 5

Variable Production and Stability of Hemolysin BL Components in Bacilluscereus

Little is known about the production kinetics and stabilities ofhemolysin BL components (B, L₁, L₂). B. cereus strains from food andnon-food sources were grown either for 16 hours at 31° C. onnitrocellulose overlaid on nutrient agar supplemented with 0.15 NaCl and2% calf serum (colony blots) or aerated for 8.5 hours at 31° C. in BHIG.The colony blots and dot blots of BHIG supernatant were probed withspecific polyclonal antibodies to each HBL component. All componentswere detected at varying levels in 99 of 102 (97%) of the colonies grown16 hours, but in only 80 (78%) of the 8.5 hour supernatants. At 8.5hours, only two of the components were detected from three strains andnone from 19 strains.

In broth cultures of three isolates, production kinetics and levelsvaried with strain, temperature, aeration and growth medium. Productionwas higher at 31° C. with aeration than 25° C. or 37° C. with or withoutaeration. Strains FM-1 and S1C produced higher levels of all componentsin BHIG than in CGY; B. cereus F837/76 produced higher levels in CGY.The stability of each components also varied. In BHIG, B and L₂increased or remained stable up to 24 hours for all strains; L₁ wasdegraded extensively for S1C and FM-1 but remained stable for F837/76.In CGY, for strains S1C and FM-1, all components were degraded rapidlybeginning after 6 hours. No components were detected for FM-1 at 24hours. For B. cereus F837/76, B and L₁ remained stable but L₂ wasslightly degraded after 8 hours. Thus, the production kinetics andstability of hemolysin BL components are highly variable.

Experiment 6

Enterotoxin Activity of Hemolysin BL From Bacillus cereus

As stated previously, hemolysin BL is a unique toxin consisting of threedifferent proteins designated B, L₁, and L₂. All three components arerequired for maximal activity in all of the biological assays tested sofar. It was previously categorized as a diarrheal toxin based on itsability to cause vascular permeability in rabbit skin. Here, HBL wastested for the ability to cause fluid accumulation in the ligated rabbitileal loop assay, the definitive assay for B. cereus diarrheal activity.Samples were injected into ileal loops in 4 New Zealand white rabbits(approximately 1 kg) per experiment. After 5 to 6 hours, fluidaccumulation was measured and activity recorded as V/L (the mean offluid volume to loop-length ratios). Differences in means were analyzedvia one-tailed t test. Compared with negative controls (V/L=0.33),injection of all three components caused significant fluid accumulationat 5 ug per component (V/L=0.59, P<0.005) and 25 ug per component(V/L=0.99, P<0.0005). V/L was not significantly different for individualcomponents or binary combinations versus negative controls (P>0.05) andwere below the 5 ug dose of all three components (P<0.05). Combinedantisera to B and L₂ completely inhibited ileal loop activity of B.cereus F837/76 culture supernatant. Preimmune serum had no effect on theactivity of the supernatant. Hemolysin BL has all of the biologicalcharacteristics ascribed to the B. cereus enterotoxin, and is 20 to 100times more potent than previously reported enterotoxin preparations.

It is understood that the present invention is not limited to theparticular reagents, steps or methods disclosed herein. Instead itembraces all such modified forms thereof as come within the scope of theclaims following the Bibliography.

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    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 3                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (v) FRAGMENT TYPE: N-terminal                                                 (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Bacillus cereus                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       SerGluIleGluGlnThrAsnAsnGlyAspThrAlaLeu                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (v) FRAGMENT TYPE: N-terminal                                                 (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Bacillus cereus                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       XaaGluThrIleAlaGlnGluGlnLysValGlyAsnTyrAlaLeuGly                              151015                                                                        ProGlu                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (v) FRAGMENT TYPE: N-terminal                                                 (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Bacillus cereus                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GluThrGlnXaaGluAsnMetAspIleXaaSer                                             1510                                                                          __________________________________________________________________________

What is claimed is:
 1. A purified protein "B" isolatable from Bacilluscereus and having the following properties:a. isoelectric point: 5.34;b. molecular weight: 37,800 Daltons; c. N-terminal sequence:S-E-I-E-Q-T-N-N-G-D-T-A-L (SEQ. ID. NO. 1); and d. extinctioncoefficient: 1.32.
 2. A purified protein "L₁ " isolatable from Bacilluscereus and having the following properties:a. isoelectric point: 5.33;b. molecular weight: 38,500 Daltons; c. N-terminal sequence:x-E-T-I-A-Q-E-Q-K-V-G-N-Y-A-L-G-P-E (SEQ. ID. NO. 2), where x isundetermined; and d. extinction coefficient: 1.85.
 3. A purified protein"L₂ " isolatable from Bacillus cereus and having the followingproperties:a. isoelectric point: 5.33; b. molecular weight: 43,200Daltons; c. N-terminal sequence: E-T-Q-x-E-N-M-D-I-x-S (SEQ. ID. NO. 3),where x is undetermined; and d. extinction coefficient: 0.83.
 4. Apurified enterotoxin of Bacillus cereus comprising the followingproteins having the following properties:B: a) isoelectric point: 5.34;b) molecular weight: 37,800 Daltons; c) N-terminal sequence:S-E-I-E-Q-T-N-N-G-D-T-A-L (SEQ. ID. NO. 1); and d) extinctioncoefficient: 1.32; L₁ : a) isoelectric point: 5.33; b) molecular weight:38,500 Daltons; c) N-terminal sequence:x-E-T-I-A-Q-E-Q-K-V-G-N-Y-A-L-G-P-E (SEQ. ID. NO. 2), where x isundetermined; and d) extinction coefficient: 1.85; L₂ : a) isoelectricpoint: 5.33; b) molecular weight: 43,200 Daltons; c) N-terminalsequence: E-T-Q-x-E-N-M-D-I-x-S (SEQ. ID. NO. 3), where x isundetermined; and d) extinction coefficient: 0.83.
 5. A purifiedbioreagent suitable for antibody assays comprising a pure protein havingthree subunits designated B, L₁, and L2, the subunits having thefollowing properties:B: a) isoelectric point: 5.34; b) molecular weight:37,800 Daltons; c) N-terminal sequence: S-E-I-E-Q-T-N-N-G-D-T-A-L (SEQ.ID. NO. 1); and d) extinction coefficient: 1.32; L₁ : a) isoelectricpoint: 5.33; b) molecular weight: 38,500 Daltons; c) N-terminalsequence: x-E-T-I-A-Q-E-Q-K-V-G-N-Y-A-L-G-P-E (SEQ. ID. NO. 2), where xis undetermined; and d) extinction coefficient: 1.85; L₂ : a)isoelectric point: 5.33; b) molecular weight: 43,200 Daltons; c)N-terminal sequence: E-T-Q-x-E-N-M-D-I-x-S (SEQ. ID. NO. 3), where x isundetermined; and d) extinction coefficient: 0.83.